Peripheral nerve injury leads to severe neuropathic pain. Previous studies have highlighted the beneficial effects of physical exercise on alleviating neuropathic pain. Exercise regulating transforming growth factor-1 (TGF-1) can improve several diseases and relieve neuropathic pain induced by peripheral nerve injury. Here, we investigated whether exercise could alleviate neuropathic pain by modulating TGF-1 expression. We assessed mechanical and cold pain behavior and conducted molecular evaluation of the spinal cord. We found that spared nerve injury (SNI) led to mechanical and cold allodynia in the hind paw, elevated the expression of latency-associated peptide- (LAP-) TGF-1, and activated astroglial in the spinal cord. Exercise decreases allodynia, astroglial activation, and LAP-TGF-1 in SNI mice. Intrathecal injection of a TGF-type I receptor inhibitor attenuated exercise analgesia and enhanced astroglial activation. These findings demonstrate that exercise induces analgesia by promoting TGF-1 activation and inhibiting astrogliosis. Our study reveals a new underlying mechanism for exercise-attenuated neuropathic pain in the maintenance stage of neuropathic pain after nerve injury.